Rhamnose binding protein

ABSTRACT

An isolated RBP with at least one of the following characteristics:
         a) a molecular weight of approximately 65-70 kDa and more preferably 66-69 kDa;   b) a pI of greater than 10 or less than 3;   c) a dissociation constant of approximately 1.5×10 −6  when bound to the rhamnose moiety of solamargine;   d) adapted to bind to a rhamnose affinity column prepared according to example 1 and under the conditions set out therein;   e) adapted to be eluted from the column in example 1 with a 100 mM rhamnose solution;   f) insoluble in aqueous solution; and   g) soluble in highly denaturing buffers containing greater that approximately 2% surfactant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/359,873, filed on Feb. 7, 2003, now U.S. Pat. No. 6,930,171 whichclaims the benefit of U.S. Provisional Application Ser. No. 60/355,593,filed on Feb. 7, 2002, which applications are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to an isolated rhamnose binding protein(RBP) that is over expressed in cancer cells relative to non-cancercells. The present invention also relates to methods of diagnosingcancer by detecting RBP levels and to RBP agonists such as antibodiesand methods of treating and diagnosing cancer using RBP agonists.

BACKGROUND ART

BEC® is a mixture of the triglycosides: solasonine and solamargine thathas anti-cancer activity. Studies on the mode of action of BEC® indicatethat the glycosides gain entry to cancer cells via a cell surfacereceptor and that the in vitro toxicity of BEC® to cancer cells isreduced by co-administration of rhamnose.

The presence of endogenous endocytic ligand receptors (EEL) has been anarea of clinical research for over 2 decades. The first EEL to beidentified was the asialoglycoprotein receptor on mammalian hepatocyteswith specificity for galactose (Ashwell & Hardford 1982). Since thistime other hepatic receptors have been identified. For example, fucose(Lehrman et al 1986), GalNAc (Kolb-Bachofen etal 1984), as well as anumber of cell receptors identified by Cramer and Gabius (1991). EEL'smay be involved in cellular recognition, cell adhesion or substratebinding.

To date no one has isolated and/or characterised the cell surfacereceptor that is central to BEC®'s mode of action. The present inventionseeks to overcome or at least partially alleviate this problem.

SUMMARY OF THE INVENTION

The present invention provides an isolated RBP with at least one of thefollowing characteristics:

-   -   (a) a molecular weight of approximately 65-70 kDa and more        preferably 66-69 kDa;    -   (b) a pI of greater than 10 or less than 3;    -   (c) a dissociation constant of approximately 1.5×10⁻⁶ when bound        to the rhamnose moiety of solamargine;    -   (d) adapted to bind to a rhamnose affinity column prepared        according to example 1 and under the conditions set out therein;    -   (e) adapted to be eluted from the column in example 1 with a 100        mM rhamnose solution;    -   (f) insoluble in aqueous solution; and    -   (g) soluble in highly denaturing buffers containing greater that        approximately 2% surfactant.

The ability of the RBP to bind ligands such as rhamnose to a RBP bearingcell, such as a carcinoma, render it useful in various methods. Forexample, it has been found that when the RBP binds a ligand, such asrhamnose, cell adhesion of the RBP bearing cells is inhibited. Thus, thepresent invention also provides a method of inhibiting cell adhesionbetween RBP bearing cells comprising the step of contacting the RBPbearing cells with an effective amount of a RBP ligand. The effectiveamount may be varied depending on the circumstances and may bedetermined by those skilled in the art. However, when the RBP ligand isrhamnose the effective amount may be approximately 70 picograms/cell.

Upon binding of a ligand to a cell associated RBP of the presentinvention, depending on the ligand, the ligand may be internalised inthe cell or remain on the cell surface. Whether or not a ligand isinternalised after binding to a cell associated RBP of the presentinvention depends on a variety of factors such as the molecular weight,charge, structure and/or biological activity of the ligand.

Thus, the present invention also provides a method of delivering anagent to a RBP bearing cell comprising contacting an agent-ligandcomplex with the RBP bearing cell.

The agent may be delivered to the cell surface or inside the cell byselecting an appropriate ligand-agent complex. For example, by selectingan agent-complex of a certain molecular weight or structure it ispossible to control the delivery of the agent to the cell surface or theinside of the cell. In this regard, it has been found that if the agentis above a certain threshold weight then it cannot be efficientlyinternalised in by the RBP bearing cell and will remain at the cellsurface.

The ability of RBP to bind ligands and either internalise or retain themon a cell surface, means the RBP may be utilised to locate and identifyRBP bearing cells. Thus, the present invention also provides a method ofdetecting a RBP bearing cell comprising the steps of: (i) contacting acell or tissue sample with an agent adapted to selectively bind to RBPand (ii) detecting the RBP bearing cells.

The agent may be varied and includes antibodies and other ligands oragonists that are adapted to bind to RBP. Furthermore, to ease detectionof the RBP bearing cells the agent may be adapted to be visualised.

Thus, the RBP of the present invention may be used to identify agentsthat bind to the RBP and thus can be used in assays for the RBP, asdiagnostics to identify RBP bearing cells or to target therapeuticagents to cancer cells via the RBP. The RBP may also form a component ofa screening system for antagonists or agonists of agents that bind tothe RBP.

These and additional uses for the reagents described herein will becomeapparent to those of ordinary skill in the art upon reading thisspecification.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: depicts a PAGE gel containing fractions 1-9 eluted from abiotinylated rhamnose-ITC affinity column using 100 mM free rhamnose andlane 10 contains standard molecular weight markers;

FIG. 2A: is a fluoro-image of proteins crosslinked to FRITC and analysedon a 4-20% polyacrylamide gel. Standards (tagged with assorted coloureddyes hence some visible by fluoro-imaging); Sample: 1) 5 μM FRITC (Batch1)+100 μM CDI; 2) 5 μM FRITC+500 μM CDI; 3) 5 μM FRITC+10 mM CDI; 4) 5μM FRITC (Batch 2)+100 μM CDI; 5) No FRITC+100 μM CDI; 6) No FRITC+500μM CDI;

FIG. 2B: is the total proteins from the gel depicted in FIG. 2A stainedwith Coomassie brilliant blue;

FIG. 3: is a graph used to calculate the molecular mass of the proteinsin FIG. 2A;

FIG. 4: is an image of A2058 cells following incubation with 12 μMfluorescein rhamnose-ITC at 37° C. for 15 min in HEPES buffered salinecontaining 2 mM Ca²⁺ and Mg²⁺;

FIG. 5: is a plot of the relationship between dose per cell at LD₅₀ andthe Day 1 cell density for each cell line;

FIG. 6: depicts the data in FIG. 5 condensed and fitted to a singleexponential function;

FIG. 7: is a plot of the relationship between dose per cell at LD₅₀ andthe Day 1 cell density for two particular breast cancer lines;

FIG. 8: is a comparison of the plots in FIGS. 6 and 7;

FIG. 9: is table containing single point LD50 data from another 11carcinomas;

FIG. 10: is a graphical representation of the data presented in thetable in FIG. 9; and

FIG. 11: illustrates the protective effects of rhamnose whenco-administered with BEC® via a graph of % cell (A2058, 600 cells)survival v's concentration of BEC®;

FIG. 12: illustrates the protective effects of rhamnose whenco-administered with BEC® via a graph of % cell (A2058, 5000 cells)survival v's concentration of BEC®;

FIG. 13 illustrates a fluoro-image of A2058 proteins crosslinked toFRITC, solvent extracted and analysed on a 4-20% SDS polyacrylamide gel.From left, Lane 1: Standards 83, 42.3, 32.2, 18.8 kD; Lane 2 blank;Lanes 3-8: replicate flasks of cells+approx 5 μM FRITC+100 μM carbonyldi-imidazole; and

FIG. 14 illustrates immunoprecipitation of FRITC-protein cross-linkedcomplex analysed on a 4-20% SDS polyacrylamide gel.

Total protein stain—Lane 1: Standards 206, 124, 83, 42.3, 32.2, 18.8 kD;Both); Lane 2 Protein A pre-clear (-ve); Lane 3 α-FITC antibodyprecipitation

DETAILED DESCRIPTION OF THE INVENTION

Rhamnose Binding Protein (RBP)

The present invention is based on the isolation and identification of acellular receptor of the lectin group that is more abundant onneoplastic (cancer) cells than non-cancer cells. The receptor (“RBP”) isadapted to bind and internalise rhamnose and thus represents a valuablediagnostic and therapeutic tool.

The present invention provides an isolated RBP comprising at least oneof the following characteristics:

-   -   (a) a molecular weight of approximately 65-70 kDa and more        preferably 66-69 kDa;    -   (b) a pI of greater than 10 or less than 3;    -   (c) a dissociation constant of approximately 1.5×10⁻⁶ when bound        to the rhamnose moiety of solamargine;    -   (d) adapted to bind to a rhamnose affinity column prepared        according to example 1 and under the conditions set out therein;    -   (e) adapted to be eluted from the column in example 1 with a 100        mM rhamnose solution;    -   (f) insoluble in aqueous solution; and    -   (g) soluble in highly denaturing buffers containing greater that        approximately 2% surfactant.

Throughout the specification, unless the context requires otherwise, theword “comprise” or variations such as “comprises” or “comprising”, willbe understood to imply the inclusion of a stated integer or group ofintegers but not the exclusion of any other integer or group ofintegers.

The RBP and other polypeptides of the invention may be in asubstantially isolated form. In this regard, it will be understood thatthey may be mixed with carriers or diluents that will not interfere withtheir intended purpose and still be regarded as substantially isolated.A polypeptide of the invention may also be in a substantially purifiedform, in which case it will generally comprise the polypeptide in apreparation in which at least 90%, 95%, 98% or 99% of the protein in thepreparation is a polypeptide of the invention.

Assays for Compounds that Bind RBP

The RBP of the present invention may be used in assays to identifycompounds that interact with (e.g., bind to) it.

The compounds which may be screened in accordance with the inventioninclude, but are not limited to peptides, antibodies and fragmentsthereof, and other organic compounds (e.g., peptidomimetics) that bindto the RBP and either mimic the activity triggered by the naturalligand—rhamnose (i.e., agonists) or inhibit the activity triggered bythe natural ligand—rhamnose (i.e., antagonists).

Other compounds that may be screened according to the present inventionare peptides, antibodies or fragments thereof, and other organiccompounds that mimic the extra cellular domain of the RBP (or a portionthereof) and bind to and “neutralize” natural ligand such as rhamnose.

Such compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries; and combinatorial chemistry-derived molecularlibrary made of D- and/or L-configuration amino acids, phosphopeptidesincluding, but not limited to, members of random or partiallydegenerate, directed phosphopeptide libraries, antibodies (including,but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic,chimeric or single chain antibodies, and FAb, F(ab′).sub.2 and FAbexpression library fragments, and epitope-binding fragments thereof),and small organic or inorganic molecules.

Computer modelling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate RBP expression or activity. Having identified such a compoundor composition, the active sites or regions are identified. Such activesites might typically be ligand binding sites, such as the interactiondomains of rhamnose with RBP itself. The active site can be identifiedusing methods known in the art including, for example, from study ofcomplexes of RBP with rhamnose. In this regard, chemical or X-raycrystallographic methods can be used to find the active site by findingwhere on the factor the complexed ligand is found. Next, the threedimensional geometric structure of the active site is determined. Thiscan be done by known methods, including X-ray crystallography, which candetermine a complete molecular structure. On the other hand, solid orliquid phase NMR can be used to determine certain intra-moleculardistances.

Having determined the structure of the active site, eitherexperimentally, by modelling, or by a combination, candidate modulatingcompounds can be identified by searching databases containing compoundsalong with information on their molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a search can be manual, but is preferably computer assisted. Thesecompounds found from this search are potential RBP modulating compounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modelling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Further experimental and computer modelling methods useful to identifymodulating compounds based upon identification of the active sites ofrhamnose and RBP will be apparent to those of skill in the art.

Although described above with reference to design and generation ofcompounds that could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds thatare inhibitors or activators.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of the RBPand for treating cancer.

The compounds capable of binding RBP may also be used to identify andisolate RBP homologues. In this regard, the compounds may be used toscreen various cell types such as cancer cell types to locate variantsof the RBP that could be used to design specific therapeutic agents fortreatment of related cancers.

In vitro systems may be designed to identify compounds capable ofinteracting with (e.g., binding to) RBP (including, but not limited to,the extra cellular domain of RBP). These compounds may be useful, forexample, in modulating the activity of wild type and/or mutant RBP;elaborating the biological function of the RBP; screening for compoundsthat disrupt normal RBP interactions; or may in themselves disrupt suchinteractions.

The principle of the assays used to identify compounds that bind to theRBP involves preparing a reaction mixture of the RBP and the testcompound under conditions and for a time sufficient to allow the twocomponents to interact and bind, thus forming a complex which can beremoved and/or detected in the reaction mixture. The RBP species usedcan vary depending upon the goal of the screening assay. For example,where agonists of the natural ligand are sought, the full length RBP, ora soluble truncated RBP, e.g., in which the transmembrane or cellulardomain is deleted from the molecule, a peptide corresponding to theextracellular domain or a fusion protein comprising the RBPextracellular domain fused to a protein or polypeptide that affordsadvantages in the assay system (e.g., labelling, isolation of theresulting complex, etc.) can be utilized.

The screening assays can be conducted in a variety of ways. For example,one method to conduct such an assay involves anchoring the RBP or fusionprotein or the test substance onto a solid phase and detecting RBP/testcompound complexes anchored on the solid phase at the end of thereaction. In one embodiment of such a method, the RBP may be anchoredonto a solid surface, and the test compound, which is not anchored, maybe labelled, either directly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the RBP or testcompound and drying. Alternatively, an immobilized antibody, such as amonoclonal antibody, specific for the protein to be immobilized may beused to anchor the protein to the solid surface.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labelled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labelled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labelled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labelled or indirectlylabelled with a labelled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for RBP or thetest compound to anchor any complexes formed in solution, and a labelledantibody specific for the other component of the possible complex todetect anchored complexes.

Cell-based assays can also be used to identify compounds that interactwith RBP. To this end, cell lines that naturally express RBP such as acancer cell line selected from the group comprising: HT-29, LS174-T.AGS, 5637, A431, 786-O, Hs578Bst, CCD 18Lu, HeLa 229, HepG2, JAM, NO36,U87-MG, DV145, LNCaP and A2058, or cell lines (e.g., COS cells, CHOcells, fibroblasts, etc.) that have been genetically engineered toexpress RBP (e.g., by transfection or transduction of RBP DNA) can beused. Interaction of the test compound with, for example, theextracellular domain of RBP expressed by the host cell can be determinedby comparison or competition with native rhamnose.

Diagnostics

The RBP of the present invention and agonists thereof can be employedfor the diagnostic and prognostic evaluation of cancer. Such methodsmay, for example, utilize reagents such as the antibodies describedherein. Specifically, such reagents may be used, for example, to detectan over-abundance of RBP relative to normal cells.

Thus, the present invention provides a method for detecting cancer in asample comprising the steps of: (i) detecting the level of RBP in thesample; and (ii) comparing it to the level of RBP in a sample from anon-cancer source.

The detection method of the present invention may be used to diagnosecancer in vitro. Thus, the present invention provides a method ofdiagnosing cancer in a patient comprising the steps of: (i) detectingthe level of RBP in a sample from the patient; and (ii) comparing it tothe level of RBP in a sample from a non-cancer source.

Alternatively, the detection method may be used to diagnose cancer invivo. In this regard, agents that are adapted to bind to RBP can belabelled and administered to a subject suspected of having cancer andlater detected to perform the diagnosis. Thus, the present inventionalso provides a method of diagnosing cancer in a patient comprising thesteps of: (i) detecting the level and/or distribution of RBP in thepatient; and (ii) analysing the distribution and/or levels of RBP toidentify differences that are indicative of cancer.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specific RBPantibody reagent described herein, which may be conveniently used, e.g.,in clinical settings, to diagnose patients suspected of having cancer.

RBP antibodies and other agonists of RBP may be used as cancerdiagnostics and prognostics, as described herein. Such diagnosticmethods may be used to detect abnormalities in the level of RBP and maybe performed in vivo or in vitro, such as, for example, on biopsytissue.

For example, antibodies directed to epitopes of the RBP can be used invivo to detect the pattern and level of expression of the RBP in thebody. Such antibodies can be labelled, e.g., with a radio-opaque orother appropriate compound and injected into a subject in order tovisualize binding to the RBP expressed in the body using methods such asX-rays, CAT-scans, or MRI. Labelled antibody fragments, e.g., the Fab orsingle chain antibody comprising the smallest portion of the antigenbinding region may also be used for this purpose. When interpreting thepatterns produced according to the diagnostic method, account must betaken on background signal or “noise” from non-cancer cells that alsobear the RBP, albeit at lower levels. However, those skilled in the artare readily able to discern noise from actual signal in performing thediagnosis. Immunoassays or fusion protein detection assays can also beused to diagnose or type cancer in biopsy or autopsy samples in vitro.

Agonists described herein including antibodies, or fragments ofantibodies may also be used to quantitatively or qualitatively detectthe presence of RBP or conserved variants or peptide fragments thereof.This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labelled antibody coupled with lightmicroscopic, flow cytometric, or fluorimetric detection.

The agonists such as antibodies (or fragments thereof) of the presentinvention may, additionally, be employed histologically, as inimmunofluorescence, immunoelectron microscopy or non-immuno assays, forin situ detection of RBP or conserved variants or peptide fragmentsthereof.

In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labelled antibody orfusion protein of the present invention. The antibody (or fragment) orfusion protein is preferably applied by overlaying the labelled antibody(or fragment) onto a biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of the RBP,or conserved variants or peptide fragments, but also its distribution inthe examined tissue. Using the present invention, those of ordinaryskill will readily perceive that any of a wide variety of histologicalmethods (such as staining procedures) can be modified in order toachieve such in situ detection.

Immunoassays and non-immunoassays for RBP or conserved variants orpeptide fragments thereof will typically comprise incubating a sample,such as a biological fluid, a tissue extract, freshly harvested cells,or lysates of cells which have been incubated in cell culture, in thepresence of a detectably labelled antibody capable of identifying RBP orconserved variants or peptide fragments thereof, and detecting the boundantibody by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support that is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labelled RBP antibody or otheragonist. The solid phase support may then be washed with the buffer asecond time to remove unbound antibody. The amount of bound label onsolid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

With respect to antibodies, one of the ways in which the antibody can bedetectably labelled is by linking the same to an enzyme. This thenrenders the antibody suitable for use in an enzyme immunoassay (EIA).The enzyme that is bound to the antibody will react with an appropriatesubstrate, preferably a chromogenic substrate, in such a manner as toproduce a chemical moiety that can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alphaglycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby calorimetric methods that employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labelling the antibodies,antibody fragments or other agonists, it is possible to detect RBPthrough the use of a radioimmunoassay (RIA). The radioactive isotope canbe detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody or other agonist with afluorescent compound. When the fluorescently labelled antibody isexposed to light of the proper wave length, its presence can then bedetected due to fluorescence. Among the most commonly used fluorescentlabelling compounds are fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

Agonists such as antibodies can also be detectably labelled usingfluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the antibody using such metalchelating groups such as diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

The antibody or other agonist can also be detectably labelled bycoupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged antibody is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Examples of particularly useful chemiluminescent labellingcompounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody orother agonist of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labelling are luciferin, luciferase and aequorin.

Methods of Treatment

The ability of the agonists of the present invention bind to RBP andsubsequently become internalised in the target cell renders them usefulfor preferentially delivering agents to cells with a higher load of RBP,such as cancer cells.

For therapeutic purposes, the agents linked to the agonists of thepresent invention may be any agent that is adapted to prevent cellgrowth or division or cause cell death such as, Doxorubicin,Daunorubicin, Vincristine, Vimblastine, Vindesine, Methothrexate,Cytarabine, Etopside, Cisplatin, Carboplatin, 5-Fluorouracil, Bleomycin,Epirubicin, Cyproterone, Irinotecan etc. When linked to such agents theagonists of the present invention may be used to treat cancer in apatient.

Thus, the present invention provides a method of treating cancer in asubject comprising administering a therapeutically effective amount of aRBP agonist anticancer conjugate to said subject.

The agonists of the present invention may also be used to treat BEC®overdose. In this regard, if BEC® has been administered to a patient attoo high a dose, then an agonist of the present invention may beadministered to bind to the RBP of the present invention and prevent orat least reduce BEC® binding.

Thus, the present invention also comprises a method of treating BEC®overdose in a subject, the method comprising administering an effectiveamount of an RBP agonist to the subject. Agonists for use in this aspectof the invention may be varied and include RBP antibodies, rhamnose orsome other RBP ligand.

Compositions/Administration

This invention also contemplates pharmaceutical or veterinarycompositions comprising an agonist of the present invention and apharmaceutically acceptable carrier. Preferably, the compositions willfurther comprise an agent adapted to cause cell death such as aglycoside. Pharmaceutical compositions of proteineous drugs of thisinvention are particularly useful for parenteral administration, i.e.,subcutaneously, intramuscularly or intravenously. The compositions forparenteral administration may comprise a solution of the compounds ofthe invention or a cocktail thereof dissolved in an acceptable carrier,preferably an aqueous carrier, an emulsion of formulated as micelles inan appropriate carrier. A variety of aqueous carriers may be employed,e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like.These solutions are preferably sterile and generally free of particulatematter. These solutions may be sterilized by conventional, well knownsterilization techniques. The compositions may further containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents.

The concentration of the compounds of the invention in suchpharmaceutical formulation can very widely, i.e., from less than about0.1%, usually at or at least about 1% to as much as 15 or 20% by weightand will be selected primarily based on fluid volumes, viscosities,etc., according to the particular mode of administration selected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 mL sterile buffered water, and50 mg of a compound of the invention. Similarly, a pharmaceuticalcomposition of the invention for intravenous infusion could be made upto contain 250 ml of sterile Ringer's solution, and 150 mg of a compoundof the invention. Actual methods for preparing parenterallyadministrable compositions are well known or will be apparent to thoseskilled in the art and are described in more detail in, for example,Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa.

The compounds described herein can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional proteins and art-knownlyophilization and reconstitution techniques can be employed.

In situations where the agonist is non-proteineous, it may beadministered alone or in combination with pharmaceutically acceptablecarriers. The proportion of which is determined by the solubility andchemical nature of the compound, chosen route of administration andstandard pharmaceutical practice. For example, they may be administeredorally in the form of tablets or capsules containing such excipients asstarch, milk sugar, certain types of clay and so forth. They may beadministered sublingually in the form of troches or lozenges in whichthe active ingredient is mixed with fillers and binders, flavouringagents and dyes; and then dehydrated sufficiently to make it suitablefor pressing into a solid form. They may be administered orally in theform of solutions that may be injected parenterally, that is,intramuscularly, intravenously or subcutaneously. For parenteraladministration, they may be used in the form of a sterile solutioncontaining other solutes, for example, enough saline or glucose to makethe solution isotonic.

The physician or veterinarian will determine the dosage of the presenttherapeutic agents that will be most suitable and it will vary with theform of administration and the particular compound chosen, andfurthermore, it will vary with the particular subject under treatment.The physician will generally wish to initiate treatment with smalldosages substantially less than the optimum dose of the compound andincrease the dosage by small increments until the optimum effect underthe circumstances is reached. It will generally be found that when thecomposition is administered orally, larger quantities of the activeagent will be required to produce the same effect as a smaller quantitygiven parenterally. The compounds are useful in the same manner as otherserotonergic agents and the dosage level is of the same order ofmagnitude as is generally employed with these other therapeutic agents.The therapeutic dosage will generally be from 1 to 1000 milligrams perday and higher although it may be administered in several differentdosage units. Tablets containing from 5 to 100 mg. of active agent areparticularly useful.

Topical Administration

The pharmaceutical compositions of the present invention may be adaptedfor topical application to a patient.

Various topical delivery systems may be appropriate for administeringthe compositions of the present invention depending upon the preferredtreatment regimen. Topical formulations may be produced by dissolving orcombining the agonist of the present invention in an aqueous ornonaqueous carrier. In general, any liquid, cream, or gel, or similarsubstance that does not appreciably react with the agonist or any otherof the active ingredients that may be introduced into the compositionand which are non-irritating are suitable. Appropriate non-sprayableviscous, semi-solid or solid forms can also be employed that include acarrier compatible with topical application and have a dynamic viscositypreferably greater than water.

Suitable formulations are well known to those skilled in the art andinclude, but are not limited to, solutions, suspensions, emulsions,creams, gels, ointments, powders, liniments, salves, aerosols,transdermal patches, etc, which are, if desired, sterilized or mixedwith auxiliary agents, e.g., preservatives, stabilizers, emulsifiers,wetting agents, fragrances, colouring agents, odour controllers,thickeners such as natural gums etc. Particularly preferred topicalformulations include ointments, creams or gels.

Ointments generally are prepared using either (1) an oleaginous base,i.e., one consisting of fixed oils or hydrocarbons, such as whitepetroleum or mineral oil, or (2) an absorbent base, i.e., one consistingof an anhydrous substance or substances which can absorb water, forexample anhydrous lanolin. Customarily, following formation of the base,whether oleaginous or absorbent, the active ingredient is added to anamount affording the desired concentration.

Creams are oil/water emulsions. They consist of an oil phase (internalphase), comprising typically fixed oils, hydrocarbons and the like,waxes, petroleum, mineral oil and the like and an aqueous phase(continuous phase), comprising water and any water-soluble substances,such as added salts. The two phases are stabilised by use of anemulsifying agent, for example, a surface active agent, such as sodiumlauryl sulfite; hydrophilic colloids, such as acacia colloidal clays,veegum and the like. Upon formation of the emulsion, the agonist iscustomarily added in an amount to achieve the desired concentration.

Gels comprise a base selected from an oleaginous base, water, or anemulsion-suspension base. To the base is added a gelling agent thatforms a matrix in the base, increasing its viscosity. Examples ofgelling agents are hydroxypropyl cellulose, acrylic acid polymers andthe like. Customarily, the agonist is added to the formulation at thedesired concentration at a point preceding addition of the gellingagent.

The amount of compound incorporated into a topical formulation is notcritical; the concentration should be within a range sufficient topermit ready application of the formulation to the affected tissue areain an amount that will deliver the desired amount of agonist to thedesired treatment site.

The customary amount of a topical formulation to be applied to anaffected tissue will depend upon an affected tissue size andconcentration of the agonist in the formulation.

In therapeutic applications, compositions of the invention areadministered to a subject afflicted with cancer in an amount sufficientto at least improve the condition of the patient and preferably cure thepatient of cancer.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician or veterinarian. In any event, the composition of theinvention should provide a quantity of the compounds of the inventionsufficient to effectively treat the cancer in the subject.

Antibodies

Antibodies that specifically recognize one or more epitopes of RBP, orepitopes of conserved variants of RBP, or peptide fragments of the RBPare also encompassed by the invention. Such antibodies include but arenot limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′).sub.2 fragments, fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above.

The antibodies of the invention may be used, for example, in thedetection of the RBP in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of RBP. Such antibodies mayalso be utilized in conjunction with, for example, compound screeningschemes, as described herein for evaluating the effect of test compoundson the ability of RBP to bind its ligand. Additionally, such antibodiesmay be used to inhibit RBP activity that may be useful in variousstudies on the dynamics of the binding between the RBP and its ligand.

For the production of antibodies, host animals may be immunized byinjection with the RBP or an immunogenic portion thereof such as onecorresponding to a functional domain of the RBP, e.g. the extracellulardomain. Host animals may include but are not limited to rabbits, mice,and rats, to name but a few.

Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminium hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Monoclonal antibodies may be obtained by any technique that provides forthe production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the monoclonals of this invention may be cultivated in vitroor in vivo. Production of high titres of monoclonals in vivo makes thisthe presently preferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine monoclonal and a human immunoglobulinconstant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to produce single chainantibodies against the RBP. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′).sub.2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′).sub.2fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

Antibodies to the RBP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” the RBP, using techniques wellknown to those skilled in the art. (See, e.g., Greenspan & Bona, 1993,FASEB J 7(5): 437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example, antibodies that bind to the RBP andcompetitively inhibit the binding of rhamnose to the RBP can be used togenerate anti-idiotypes that “mimic” the extracellular domain of the RBPand therefore bind rhamnose.

The present invention will now be described with reference to a numberof examples. The examples are in no way limiting on the precedingdescription.

EXAMPLES Example 1 Isolation of a Rhamnose Binding Protein UsingAffinity Chromatography

Materials/Methods

1. Labelling of Rhamnose Probes

Biotin Rhamnose-ITC (BRITC) was formed by dissolving Rhamnose-ITC (SigmaR6881; RMM 297.3) in DMSO, diluting it to 1 mg/ml in 10 mM sodiumbicarbonate pH 9.1 and then adding biotin hydrazide (Sigma, RMM 258.3)at 1:1 or 5:1 molar ratio and allowing the reaction to proceed at roomtemperature for 16 h.

2. Preparation of Rhamnose Affinity Column

Streptavidin sepharose conjugated columns (Amersham 17-5112-01) or freeresin (17-5113-01) with a theoretical capacity for biotin labelledrhamnose (BRITC) of 60 μg/ml was used. An excess amount of BRITC wasdissolved in phosphate buffered saline (PBS) and circulated over thepre-equilibrated column at a flow rate of 0.2 ml/min for 30 min.Successful coupling of the BRITC was monitored by HPLC analysis of theBRITC-PBS solution.

3. Cell Lysis

Packed, washed cells were lysed by freeze thawing (−80° C./4° C.)followed by brief sonication (30-40 sec at 50% duty pulse using 375Wsonicator fitted with microtip probe). Cells were lysed in the presenceof protease inhibitor cocktail (Roche 1-836-170) in order to minimiseproteolysis.

4. Multiple Surfactant Solution (MSS)

MSS comprises 5 M urea, 2 M thiourea, 0.002 M n-tributyl phosphine, 0.5%pH 3-10 Pharmalyte carrier ampholytes (Pharmacia, Uppsala) [only in 2-Dpreparations], 2%3-([3-cholamidopropyl]-dimethylammonio)-1-propanesulfonate (CHAPS), 2%caprylyl sulfo-betaine, and 0.001% Orange G dye. Material was alsotreated with endonuclease EC 3.1.30.2 in order to eliminatecontaminating DNA.

5. One-dimensional Polyacrylamide Gel Electrophoresis (1D-PAGE)

Pre-cast Tris-HCl 4-20% polyacrylamide gradient gels (Bio-Rad) were usedwith electrode buffer Tris/glycine, pH 8.3. Sample loading solution:Tris pH 6.8, 0.1% SDS, glycerol, dithiothreitol, bromophenol bluemarker.

Electrophoresis conditions: 100V for 90 min.

6. Protein Visualisation In-gel

This was accomplished either by staining with silver, or with CoomassieR250 in water/methanol/acetic acid. Fluorescence was visualised using aFluoro-imager (Pharmacia).

7. Affinity Chromatography

Whole cell lysis preparations were prepared using MSS on 10⁸-10⁹ A2058cells. The solubilised protein was then diluted 1/50 into HEPES bufferedsaline containing 140 mM NaCl, 2 mM MgCl₂, and 2 mM CaCl₂, pH 7.4(HBS²⁺) and passed sequentially over a control column (no rhamnose) andthe rhamnose affinity column. Each column was washed with HBS²⁺ andeluted with 100 mM rhamnose. Fractions were analysed using acrylamidemini-gels and 1D SDS-PAGE, followed by silver staining.

Results

A band with a molecular weight of approximately 65 kD was visualised inthe eluent fractions (FIG. 1). No bands were visible in eluent from acorresponding control column that did not contain BRITC (results notshown).

Example 2 Cross-linking the Rhamnose Binding Protein and Its Ligand

Materials/Methods

(a) Labelling of Rhamnose Probes

Fluorescein Rhamnose-ITC (FRITC) was formed by reacting Rhamnose-ITCwith fluorescein amine (Sigma F1148, RMM 347.3) at a molar ratio of 1:10in 10 mM sodium bicarbonate pH 9.1.

(b) Cell Lysis

As per example 1.

(c) Multiple Surfactant Solution (MSS)

As per example 1.

(d) One-dimensional Polyacrylamide Gel Electrophoresis (1D-PAGE)

As per example 1.

(e) Protein Visualisation In-gel

As per example 1.

(f) Cell Probing

A2058 cells were coated at low density in a microscope compatiblechamber. The cells were washed with HBS²⁺ then incubated with FRITC (topconcentration of DMSO=2.5%) for 5-15 min at 37° C. The FRITC wasremoved, the cells washed twice with HBS²⁺ and examined under a visiblelight microscope. The incubation was repeated with 0-100 μMfluorescein-amine, and for 25 and 6 μM FRITC using a 10³× excess ofunlabelled rhamnose.

(g) Cell Surface Receptor Cross-Linking

A2058 cells prepared in 40 ml culture flasks were incubated with FRITC(5 or 10 μM) as described above and washed once with HBS²⁺. Carbonyldi-imidazole (Aldrich 115533) was dissolved at 1M in DMSO immediatelyprior to use. This stock solution was then diluted in HBS²⁺ or DMSO to100 μM-10 mM and added to the cells at room temperature. After 15 minthe cross-linker was removed and the flasks stored on ice. Cells werethen removed from the flasks by scraping and taken up into MSS lysisbuffer. Protein fractions were subjected to SDS-PAGE and the gelsvisualised as set out above.

(h) Two Dimensional Electrophoresis

Cross linked cells prepared according to method 7 above, using 5 μMFRITC, were lysed into 350 μl of MSS by cell scraping. This material wasprepared for iso-electric focusing by loading onto an 18 cm ImmobilineDryStrip, pH 3-10 [Amersham], the strip equilibrated and electrophoresedfor approx. 150 kVhr (Voltage gradient: 200V, 12 hr; 250V, 1.5 hr; 500V,2 hr; 1000V, 2.5 hr; 8000V, 19 hr).

The strip was then run in the second dimension using 1-D PAGE accordingto method 4 above, except a 10% polyacrylamide gel was used. The gel wasanalysed using a fluoroimager and silver stained.

Results

The electrophoretic profile of the protein components from the crosslinking experiments are set out in FIGS. 2A and 2B. FIG. 2A depicts theproteins cross-linked to fluorescein that were visualised byfluorescence scanning and FIG. 2B depicts the total protein stained withCoomassie brilliant blue.

The total protein stain indicates that there are approximately equalamounts of protein loaded in each lane. Following FRITC incubation twofluorescently labelled proteins are detectable that are not present inthe CDI only lanes. Calibration of the gel using the molecular massmarkers (FIG. 3) gives masses of 22 kD and 68 kD for these proteins.However, these masses include one or more FRITC molecules andconsequently the mass of the receptor is approximately 67 kD.

The results from the two dimensional electrophoresis suggest thefluorescein tagged protein is running with a pI of approximately 6-7,and the molecular mass is consistent with prior results.

Example 3 Staining of Cells with a Fluorescein Tagged Rhamnose Probe(FRITC)

Materials/Methods

A fluorescein tagged rhamnose probe (FRITC) was prepared as previouslydescribed and used to stain A2058 cells.

FRITC at concentrations from 3-25 μM was incubated with A2058 cells for15 minutes at 37° C.

Results

The cells following incubation were found to fluoresce confirming thepresence of a rhamnose binding protein on the cells. An image of thecells is depicted in FIG. 4 and closer inspection of the stained cellsindicates an increased concentration of staining in the cell nucleus,suggesting the rhamnose probe is also taken inside the nuclear membrane.It was found that the staining could be inhibited by co-incubation ofthe FRITC and cells with free rhamnose at 10 mM concentration.

Example 4 Effect of Cell Density on Measured LD₅₀ Values

Materials/Methods

Given that the evaluation of the cytotoxicities for different cell linesneeded to be conducted using different sized cell populations, it wasconsidered prudent to determine the effect, if any, of cell number onthe measured value of LD₅₀. Five cell lines, HT-29, LS174-T, 5637, A431and MCF-7 were evaluated at four different seeding cell densities.Hs578T and CCD 18Lu were evaluated at three seeding densities andHs578Bst, both early and late passage cells, were evaluated at twoseeding densities.

In order that the full range of cell densities be evaluated with atleast one cell line, a 3-day version of the cytotoxicity was developed.The cell lines involved were recalibtrated for the 3-day format.Multiwell plates were seeded on day 1. Cells were treated with BEC® onday 2 and MTT was added twenty four hours later.

Results

Plotting the relationship between dose per cell at LD₅₀ and the Day 1cell density for each cell line, FIG. 5, reveals that the behaviours ofthe epidermoid adenocarcinoma A431, the colorectal adenocarcinoma HT-29and the normal infant lung fibroblast line, CCD 18Lu, are identical.Similarly, the plots for the colon adenocarcinoma LS174-T and thebladder carcinoma 5637 are almost coincident.

In fact, data from CCD 18Lu, A431, HT-29, LS174T and 5637 can becombined and fitted to a single exponential function,Dose per seeded cell at LD ₅₀=Intercept×e ^(−k×Seeding density)+Limitas shown in FIG. 6.

This implies that, for these five cell lines the processes of BEC®uptake, including receptor affinity, hydrolytic processing to producethe lysogenic ligand complex, as well as the pathway to cell death, arequantitatively identical.

Similarly, data from the two breast cancer lines, Hs578T, aninfiltrating ductal carcinoma and MCF-7, a metastasis from a breastadenocarcinoma, appear to behave similarly to each other but differentlyfrom the other lines. These combined data sets can also be fitted to anexponential function, see FIG. 7.

Comparing the two fitted functions, two distinct regions are discernable(FIG. 8). The functions are virtually coincident at cell densities of1,500 cells per well and below, conditions under which receptor affinitycan be expected to be the major determinant of cytotoxicity (region 1 ofFIG. 8). This suggests that only a single type of receptor is involvedin virtually all cell lines included in this study. We estimate that thedissociation constant for this receptor is likely to be of the order of1.5×10⁻⁶ M.

However, the functions diverge at cell densities greater than 1,500cells per well (region 2 of FIG. 8). The difference between the limitvalues, representing the minimum dose per cell to kill 50% of thesusceptible population, is obvious. For LS174-T and 5637 the fittedvalue of this minimum dose is 300 pg BEC®/cell (71 pg solamargine/cell)while for the breast cancer lines this minimum dose is some three-foldhigher at 580 pg BEC®/cell (137 pg solamargine/cell). Such a differencecould arise from either a significantly lower number of receptors percell or slower intracellular processing to produce the isolated ligandcomplex.

Note from FIG. 5 that the behaviour of early passage normal breastfibroblasts differs from late passage cells of the same line. Within thelimitations of the restricted data sets these non-tumour cells appear tobecome more vulnerable to BEC® as they approach senescence.

Example 5 Single Point Data for Other Cell Lines

Materials/Methods

A range of other cell lines were assessed in a similar manner to theassessments carried out in Example 4.

Results

FIG. 10 shows that the single data points for the other cell linesevaluated are plotted, with the exception of MIA PaCa-2, the breastcancer lines and the early passage normal breast fibroblasts, all fallon the same exponential curve of FIG. 6.

Example 6 Co-administration of BEC® and Rhamnose

Materials/Methods

A2058 cells at two different cell densities were treated with BEC®+/−rhamnose and cell survival was monitored after 4 days. The treatmentscomprised: (i) BEC® only for 4 days (ii) BEC® only for 5 minutes (iii)BEC® and 5 mM rhamnose for 4 days and (iv) BEC® and 5 mM rhamnose for 5minutes.

Results

FIGS. 11 and 12 indicate that rhamnose competition with BEC® uptake ismore readily observed at the higher cell density (5000 cells). Underthese conditions, where the amount of BEC® available to each cell is amajor factor determining LD₅₀, the presence of rhamnose at therelatively high concentration of 5 mM affects the amount of BEC® takenup by the cells in both 5 minutes and 4 days from solutions in specificconcentration ranges. Data in FIG. 12 suggests that the rhamnoseprotective effect is more significant in the pulsed treatmentexperiment.

Example 7 Isolation of RBP

Materials/Methods

1. Preparation of FRITC

The following reaction mixture was used:

Rhamnose  5 mg/ml in 500 μl DMSO Isothiocyanate [Sigma R6881]Fluorescein [Sigma  50 mg in 500 μl F1148] 100 mM sodium 120 μl hydrogencarbonate MilliQ water  80 μl

Incubate overnight at room temperature and store at −20° C. Purify FRITCby RP-HPLC:

-   -   Column: C18 reverse phase    -   Solvent A: water+0.06% tri-fluoro acetic acid (TFA)    -   Solvent B: 80% acetonitrile (ACN)/water+0.06% TFA    -   Analytical gradient 98-50% (A) over 15 min. Flow 1 ml/min.        Wavelength 255 nm or 220 nm.    -   Preparative gradient 98% (A) for 2.5 min., 98-50% (A) over 20        min. Flow 2 ml/min. Wavelength 255 nm or 220 nm.        2. Cell Surface Receptor Cross-Linking

A2058 cells were grown to 80-90% confluency in 25 ml culture flasks andthen washed with 2×HEPES buffered saline containing 140 mM NaCl, 2 mMMgCl₂, and 2 mM CaCl₂, pH 7.4 (HBS²⁺). FRITC was dissolved in a minimalvolume of DMSO (5-10 μl) and added to 1 ml HBS²⁺ to give a concentrationof 2-10 μM FRITC. The FRITC solution was added to the cells and theflask incubated for 15 min at 37° C. The FRITC was removed, the cellswashed once with HBS²⁺, and freshly prepared carbonyl di-imidazole (100μM in 1 ml DMSO) was added immediately at room temperature. After aminimum of 15 min the cross-linker was removed and the flasks stored onice.

3. Protein Extraction Following Receptor Cross-linking

All the DMSO is aspirated from the cells and 300-400 μl of MSS (section10) is added to the flask. The MSS is spread over the entire surfacearea of the flask and the cells then scraped using a cellscraper/harvester. The cells in MSS are allowed to incubate for 15 minat room temperature to remove as much of the protein as possible.

Cell extracts are removed from the flask and added to a fresh tube. Theprotein is precipitated by adding 1 ml of methanol (or acetone) andstoring the sample over night at −80° C. To remove viscous material(e.g. DNA, lipid etc.) the tube was centrifuged for 30 min at 13000 rpmand 4° C. The methanol was removed and the sample resuspended in 300 μlof MSS. To this 1.2 ml of hexane was added and the sample againcentrifuged for 30 min at 13000 rpm and 4° C. The top layer wasdiscarded and any white particulate matter on the surface of the aqueouslayer was also removed. Samples were then analysed by SDS-PAGE.

4. Immunoprecipitation of FRITC Cross-linked Proteins

100 μl of protein extract (section 1 above) was diluted to 10 ml with 50mM Tris-Cl pH 7 and 0.05% Tween 20. In order to pre-clear non-specificbinding material 50 μl of protein A sepharose [Amersham] was added andincubated overnight at 4° C. on a rotating wheel. After incubation, thesample was centrifuged at 2000 rpm for 5 min to pellet the sepharose.The supernatant was added to 10 μl of anti-FITC antibody [Sigma], 50 μlof protein A sepharose and incubated overnight at 4° C. on a rotatingwheel. After incubation the sepharose was washed 2×1 ml with 10 mM TrispH 7 and the whole sample (matrix included) was run on 4-20% SDS-PAGE.The gel was then assessed for FRITC labelled proteins by fluorescentdetection.

5. 1D PAGE was carried out as in Example 1.

6. Protein Visualisation In-gel

To detect fluorescently labelled proteins gels were scanned using aFluoro-imager (Pharmacia): fluorescein excitation wavelength 494 nm,emission wavelength 520 nm.

Visual staining was accomplished either with Coomassie G250 inwater/methanol/acetic acid or silver staining (PI in-house massspectrometry compatible protocol; under optimised conditions this isapproximately 10-fold less sensitive than previously used methods).

Results

1. Preparation of FRITC (Fluorescein Rhamnose Iso-thiocyanate)

Large quantities of fluorescein labelled rhamnose probe (FRITC) werepurified by HPLC and their viability confirmed by mass spectrometry. Theprobe appears stable indefinitely if stored dry at −20° C.

2. Cell Surface Receptor Cross-Linking

Cross linking of FRITC to the surface of A2058 cells was performedsuccessfully in 25 ml culture flasks. Large scale FRITC cross-linking(75 ml flasks) using identical concentrations of reagent wasunsuccessful. This suggests the reaction is readily influenced bymicro-changes in the cell environment. Our observations also indicatedthat an advantageous side effect of using carbonyl di-imidazole crosslinker was that the cells became adhered to the flask surface during theprocedure and hence were easier to wash.

A cross-linked FRITC protein complex was consistently visible onSDS-PAGE gels by fluorescent imaging (FIG. 13).

3. Protein Extraction Following Receptor Cross-linking

Our procedures have focused on maximising the yield of theFRITC-receptor complex, and subsequently isolating the complex fromunwanted contaminants. The extraction procedures have involved differentprotein precipitation methods and subsequent solubilisation steps. TheA2058 Receptor-FRITC complex appears fully soluble in multiplesurfactant solution (SPRL21111), and partially soluble in a range ofnon-ionic detergents (2% Tween 20, 2% Triton X-100, 2% CHAPS), howeverno single non-ionic detergent has been identified that fully solubilisesthe complex.

Further, the fluorescent complex appears to be associated with the celldebris/DNA that is precipitated during the initial methanolprecipitation. To overcome this problem (of contamination and viscosity)we have developed a two-stage clean-up using methanol, followed byhexane.

4. Immunoprecipitation of FRITC Cross-linked Proteins

The protein-FRITC complex was diluted into a low detergent, low saltbuffer and incubated with an antibody directed against fluorescein. Anycomplexes formed were absorbed onto protein A, precipitated and analysedby SDS-PAGE. The experiments produced a feint protein band at approx 70kD that was detectable by Coomassie blue staining FIG. 14).

Further modifications and adaptations not specifically disclosed hereinthat are apparent to those skilled in the art upon reading thisspecification are encompassed within the scope of this invention.

REFERENCES

-   1. Ashwell, G and Harford, J. (1982) “Carbohydrate specific    receptors of the Liver”. Ann Rev. Biochem, 51, 531-554.-   2. Lehrman, M A. et al (1986) “The binding of fucose containing    glycoproteins by hepatic lectins”. J. Biol. Chem., 261 (16)    7412-7418.-   3. Kolb-Bachofen, V. et al (1984) “Gal/NAC/Gal specific rat liver    lectins their role in cellular recognition”. Biol. Cell, 51,    219-226.-   4. Cramer, F. and Gabius, H J. (1991) U.S. Pat. No. 5,225,542.

1. A method for detecting cancer in a sample comprising the steps of:(i) detecting the level of rhamnose binding protein (RBP) in the sample;and (ii) comparing it to the level of RBP in a sample from a non-cancersource, wherein said RBP has the following characteristics: i. the RBPhas a molecular weight of about 65 kDa to about 70 kDa as determined bySDS-PAGE; ii. the RBP is insoluble in aqueous solution; iii. the RBP isadapted to bind to a rhamnose affinity column in the form of astreptavidin sepharose conjugated column or free resin with atheoretical capacity for biotin rhamnose ITC (BRITC) of 60 μg/ml,wherein the BRITC is coupled to the column by passing a phosphatebuffered solution comprising an excess of BRITC dissolved therein overthe column at a flow rate of about 0.2 ml/min for 30 minutes; and iv.the RBP is adapted to be eluted from said rhamnose affinity column witha 100 mM rhamnose solution.
 2. A method of diagnosing cancer in apatient comprising the steps of: (i) detecting the level of RBP in asample from the patient; and (ii) comparing it to the level of RBP in asample from a non-cancer source, wherein said RBP has the followingcharacteristics: i. the RBP has a molecular weight of about 65 kDa toabout 70 kDa as determined by SDS-PAGE; ii. the RBP is insoluble inaqueous solution; iii. the RBP is adapted to bind to a rhamnose affinitycolumn in the form of a streptavidin sepharose conjugated column or freeresin with a theoretical capacity for biotin rhamnose ITC (BRITC) of 60μg/ml, wherein the BRITC is coupled to the column by passing a phosphatebuffered solution comprising an excess of BRITC dissolved therein overthe column at a flow rate of about 0.2 ml/min for 30 minutes; and iv.the RBP is adapted to be eluted from said rhamnose affinity column witha 100 mM rhamnose solution.
 3. A method of diagnosing cancer in apatient comprising the steps of: (i) detecting the level and/ordistribution of RBP in the patient; and (ii) analyzing the distributionand/or levels of RBP to identify differences that are indicative ofcancer, wherein said RBP has the following characteristics: i. the RBPhas a molecular weight of about 65 kDa to about 70 kDa as determined bySDS-PAGE; ii. the RBP is insoluble in aqueous solution; iii. the RBP isadapted to bind to a rhamnose affinity column in the form of astreptavidin sepharose conjugated column or free resin with atheoretical capacity for biotin rhamnose ITC (BRITC) of 60 μg/ml,wherein the BRITC is coupled to the column by passing a phosphatebuffered solution comprising an excess of BRITC dissolved therein overthe column at a flow rate of about 0.2 ml/min for 30 minutes; and iv.the RBP is adapted to be eluted from said rhamnose affinity column witha 100 mM rhamnose solution.
 4. A method according to any one of claims1, 2 and 3, wherein the level of RBP is detected using an antibody or afragment thereof.
 5. A method according to claim 4, wherein the antibodyor fragment thereof is selected from the group consisting of: apolyclonal antibody, a monoclonal antibody, a humanized or chimericantibody, a single chain antibody, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above.
 6. Amethod according to claim 4, wherein the antibody or fragments thereofis labeled.
 7. A method according to claim 6, wherein the antibody islabel is radioactive, fluorescent or luminescent.
 8. A method accordingto any one of claims 1, 2 and 3 wherein said RBP is furthercharacterized in that it has a pI of greater than 10 or less than
 3. 9.A method according to any one of claims 1, 2 and 3 wherein said RBP isfurther characterized in that it has a dissociation constant of about1.5×10⁻⁶ when bound to a rhamnose moiety of solamargine.
 10. A methodaccording to any one of claims 1, 2 and 3, wherein said RBP is furthercharacterized in that it is soluble in a denaturing buffer comprising atleast about 2% surfactant.
 11. A method according to claim 8, whereinsaid RBP is further characterized in that it is soluble in a denaturingbuffer comprising at least about 2% surfactant.
 12. A method accordingto claim 9, wherein said RBP is further characterized in that it issoluble in a denaturing buffer comprising at least about 2% surfactant.13. A method according to claim 1, 2 or 3, wherein said RBP has amolecular weight of about 66 kDa to about 69 kDa.
 14. A method accordingto claim 1, 2 or 3, wherein said RBP has a molecular weight of about 67kDa.